JP2014509523A - Multifunctional bioreactor system and method for sorting and culturing cells - Google Patents

Abstract

  The present disclosure relates to bioreactors, and more specifically to bioreactors for growing and separating cells. The bioreactor comprises at least one reaction chamber, an adjustable magnetic field, a multifunctional cell assist system, and may further comprise an optional protective perfusion system and an optional computer controlled system. Good.

Description

  The present invention relates to a multifunctional bioreactor for cell culture and cell sorting.

  This application relates generally to bioreactors, and more specifically to bioreactors that grow and separate cells.

  Many types of cells, especially hematopoietic stem cells and immune cells, need to be isolated from the original sample before they can be effectively grown in culture and directly differentiated. This isolation procedure is also called cell sorting or cell separation. Previously, cell sorting and cell culture were each performed in separate systems, in which case the target cells are first isolated and then transferred to a culture vessel. This conventional method is difficult to use, has a high risk of cell contamination and cell loss from cell sorting to cell culture, and involved two completely different devices and systems. The present inventors' present invention with a new design allows these two different procedures to be completed in a single container (chamber), thus minimizing the risk of contamination and loss of target cells and operating. Efficiency is significantly increased.

  Certain cells are very sensitive to shear stress in the culture. For example, shear stress can cause non-specific differentiation and increase apoptosis in stem cell culture, significantly reducing stem cell proliferation efficiency and direct differentiation. Increasing shear stress releases more non-specific protein during protein expression, reducing the ratio of the protein of interest in culture and thus increasing protein purification. Static culture has minimal shear stress, but the cells in static culture are usually located at the bottom of the culture vessel. Yes, and therefore not suitable for large-scale cell growth. Certain bioreactors (eg, NASA's rotating wall vessel (RWV) bioreactors) were designed to reduce shear stress. However, these bioreactors must maintain the cells in suspension by continuously moving, agitating, and / or mechanically agitating the cells. When the bioreactor stops moving, cells appear to accumulate somewhere in the bottom, but are not evenly distributed and are detrimental to most cell growth. Thus, the shear stress is small in these bioreactors, but when the bioreactor is moved, this small shear stress is continuously applied to the cultured cells. In the present invention, when the bioreactor is in a stationary state, the cells can be uniformly distributed on the bottom of the culture chamber or the surface of the magnetic beads. Thus, the present invention provides the best growth conditions for cells both in suspension and at rest.

  Some bioreactors use magnetic elements (specifically blades or buns) that are controlled to mechanically agitate the medium by a magnetic impeller and keep the cells in suspension. This type of bioreactor deliberately increases the shear stress for specific cell culture requirements. In addition to the different applications, the bioreactor of the present invention does not use a magnetic element blade or cane, and the magnetic bead of the present invention does not actually magnetize if the magnetic bead is not present in a magnetic field. It is not tinged and can only obtain magnetic force when placed in a magnetic field. The magnetic beads of the present invention are not controlled by the impeller, but are controlled by changes in magnetic field strength that affect the movement of the beads. An appropriate microenvironment (or called a niche) is very important for the growth of certain types of cells (eg, stem cells). Some devices use solid materials to create a niche or use a gel-like substance to create a niche. With these devices, after cell culture, the cells need to be washed out of the niche using a specific procedure, or the material that forms the niche must be melted or digested with enzymes to release the target cells. In the present invention, the space between the beads is usually derived from a niche for cell growth, and if the beads are lifted by changing the magnetic field strength, the cells can be easily released. Other cell culture systems have attempted to build a niche with well-like culture plates (eg, common 96-well plates), fine chambers (Hung 2005) or fine sieves (Zhang 2009). The bioreactor of the present invention is superior to well-like plates because the movement of beads creates a dynamic microenvironment for cells that require increased media flow to grow. In addition, there are fewer challenges in manufacturing, it is easier to sterilize after use, and the niche size can be easily modified by adjusting the bead size, so that the fine chamber and fine sieve Better than.

  Many cell culture vessels (chambers) have been designed for use in bioreactors such as, for example, common cell culture flasks, gas permeable bags, and rotating wall vessels. These containers can be used with common perfusion systems that can dilute cells and change media. However, while these media are used to change the media, cytokines, proteins and other expensive substances and cell products for cell growth are simultaneously removed from the media. Also, the efficiency of a typical dialysis process is not high enough. The cell culture chamber designed in the present invention takes advantage of the difference in colloid osmotic force between the permeation chamber and both sides of the culture chamber, without losing a specific size of cytokines, peptides, proteins and other materials. The medium can be changed quickly through. This cell culture chamber design provides a new perfusion technique and provides a system called the gradient osmotic perfusion system.

  Most bioreactors were designed to culture either adherent cells or suspension cells. No bioreactor has yet been reported that supports the growth of partially adherent cells. The bioreactor of the present invention can be used for almost any cell, including suspension cells, adherent cells and partially adherent cells.

  It is very common to use a computer to control the movement of a bioreactor, and many bioreactors can be programmed. In the present invention, (1) the strength and direction of the magnetic field, (2) the frequency and speed at which the cell culture chamber is turned over, (3) the frequency and speed of the magnetic beads as a result of (1) and (2) above are It is emphasized that the program is controlled by a program that responds to data received from the selected program and / or detector and sends feedback back to the bioreactor.

  Similar to the use of computers in bioreactor control, several cell density detectors using certain light sources (eg, laser projectors) have been designed to monitor cell concentration during cell culture. Data obtained from the detector or sensor is used to determine whether it is necessary to dilute the cells when it is necessary to change the medium. In the bioreactor of the present invention, the cell density detector uses a general light source and uses the data specifically to adjust the strength of the magnetic field, the speed and frequency of overturning the cell culture chamber, and a magnet. Adjust the moving speed and frequency of the beads indirectly.

  Bioreactors are typically used to grow cells in culture. However, many types of cells need to be isolated from the original sample before they can be effectively grown in culture and directly differentiated. This isolation procedure is also called cell sorting or cell separation. Typically, cell sorting and cell culture are performed in separate systems, where target cells are first isolated and then transferred to a culture vessel. This conventional method is difficult to use, has a high risk of cell contamination and cell loss from cell sorting to cell culture, and involves two completely different devices and systems.

In addition, many bioreactors use rotating impellers or the like to mix the contents of the bioreactor. Unfortunately, this adds shear stress to the cells that can destroy the cells, reducing the efficiency of the system and resulting in the release of waste products (eg, non-specific protein expression). Further, in certain bioreactors, cells may aggregate at the bottom of the cell culture chamber or other locations in the cell culture chamber. Aggregation of these cells in the culture chamber hinders effective cell growth. Thus, there is a significant need for an efficient bioreactor that can grow and separate cells while also minimizing the shear stress applied to the cells.

  In one embodiment, a bioreactor system for growing and separating cells includes a cell culture chamber that includes an interior having a first portion and a second portion, an inner first portion disposed within the culture chamber, An agitator capable of moving between the inner second part and a control system coupled to the cell culture chamber and operable to move the agitator between the inner first part and the inner second part; Is provided.

  In another embodiment, a method for growing and separating a cell provides a chamber including an interior having a first portion and a second portion, and moves between an inner first portion and an inner second portion. The first part and the second part so that the agitator is capable of being placed inside, carrying the cell culture medium inside the chamber, transferring the target cells inside the chamber, and mixing the cells and medium. Moving the agitator between the portions and removing waste from the inside of the chamber while maintaining a significant amount of target cells inside the chamber.

  In yet another embodiment, the inside of the cell culture chamber is coupled to a fluid flow to and from the cell culture medium container, the first compartment is configured to receive the cell culture medium from the container, and the agitator is internal. And a second compartment disposed between the first compartment and the second compartment so that the cell culture medium can selectively flow from the first compartment to the second compartment. The first membrane is connected to the buffer container so that the fluid can go back and forth, and the buffer can be received from the buffer container, and further, the fluid can be connected to the waste container so that the fluid can go back and forth. A third compartment configured to allow waste to flow from the third container to the waste container, and disposed between the second compartment and the third compartment, and from the second compartment to the third compartment; Medium and waste into the compartment And a second film but made to be able to flow selectively.

  When considered in conjunction with the following description, the drawings are presented for the purpose of assisting in understanding the subject matter of the invention desired to be protected.

FIG. 1 is a schematic diagram showing an illustrative bioreactor. 2a-2f show a series of schematic diagrams of a bioreactor showing agitator movement within a series of chambers. FIG. 3 is a schematic isometric view of the chamber. FIG. 4 is a schematic side view of the chamber. The figure is a schematic diagram of an agitator.

  Referring to FIG. 1, a bioreactor system 10 for growing and separating cells is shown. The system 10 includes a cell culture chamber 15, an agitator 20, and a control system 30.

The cell culture chamber 15 comprises an inner side 35 for receiving and growing target cells in a cell culture medium disposed in the cell culture chamber, a first end 40 and a second end 45. As used herein, “target cell” refers to a cell that is placed in the chamber 15 and grows in the chamber 15. Although the present disclosure is given to grow target cells, it is understood that the system may be used to mix chemicals or any other suitable solution or material. right. Furthermore, although the first end 40 and the second end 45 are shown as being at the top and bottom of the chamber 15, respectively, the ends 40, 45 can be in any suitable arrangement relative to each other (eg, in a horizontal plane). It will be understood that it is within the scope of this disclosure. As described below, the chamber 15 may include one or more inner compartments. In addition, as will be apparent to those skilled in the art, the chamber 15 may comprise a rigid material, a flexible material, a combination of a rigid material and a flexible material, a gas permeable material or any other suitable material, It may be made from any suitable material. The chamber 15 may further include one or more ports for allowing fluid to flow between the chamber interior 35 and one or more containers. Illustrative containers include, but are not limited to, cell culture medium containers, waste containers, buffer containers, CO ₂ containers, or any other suitable container.

Referring now to FIG. 3, an illustrative alternative chamber 15 is shown. In this embodiment, the chamber 15 includes a first compartment 75, a second compartment 80, and a third compartment 85. The first compartment 75 includes a first compartment and one or more containers (eg, a cell culture medium container) so that media can be added to and / or removed from the first compartment. One or more inlets / outlets 18a for providing a connection to allow fluid to flow back and forth, a buffer container such that buffers can be added to and / or removed from the first compartment, waste container as may removed from compartment 75 of 1, adding CO ₂ to the first compartment, and / or CO ₂ container, such as can be extracted from the first compartment, or any other suitable container, You may have. The first compartment 75 and the second compartment 80 are separated by the first film 90. The first membrane 90 is made so that the culture medium can selectively flow from the first compartment 75 to the second compartment 80. The membrane 90 may include penetrations, openings, or any other suitable structure and / or other media so that media can flow from the first compartment 75 to the second compartment 80. Semi-permeable in style. In one embodiment, membrane 90 is a dialysis membrane. Membrane 90 may be made from any suitable material including, but not limited to, cellulose and cellophane.

  The second compartment 80 is configured to hold the agitator therein and allow the agitator to move within the compartment as previously described in connection with FIG. Target cells typically grow and remain in the second compartment 80 as well. The second compartment 80 includes one or more ports 18b to provide a connection that allows fluid to flow between one or more containers (eg, a container of the type described with respect to the first compartment 75). May be. The second section 80 and the third section 85 may be separated by the second film 95. The second membrane 95 is made such that media and / or waste can be selectively flowed from the second compartment 75 to the third compartment 85. The membrane 95 may include penetrations, openings, or any other suitable structure and / or flow media and / or waste from the second compartment 80 to the third compartment 85. Be semi-permeable in other ways as possible. In one embodiment, membrane 95 is a dialysis membrane. Membrane 95 may be made from any suitable material including, but not limited to, cellulose and cellophane. The third compartment 85 is 1 for providing a connection that allows fluid to flow between the third compartment 85 and one or more containers (eg, a container of the type described with respect to the first compartment 75). Two or more doorways 18c may be provided. Further, in at least one embodiment, at least a portion of the outside of the chamber may be made from a gas permeable material. If at least a portion of the outside of the chamber is made from a gas permeable material, the system is configured to maintain direct gas injection into the chamber and the pH of the medium, if any, but not all Contains no elements. The first compartment 75 and the third compartment 85 may be located at any suitable location relative to the second compartment 80, and the present disclosure may be arranged in any manner with the first compartment 75 and the third compartment 85. It is not limited to the 2nd division 80 arrange | positioned between these divisions 80. FIG. Further, it will be appreciated that any suitable number of compartments may be used and the present disclosure is not limited to only three compartments.

In one embodiment, a medium containing a suitable solution containing a relatively small amount of large molecules and / or large polar molecules is provided in the first compartment 75 and has the same colloid permeability for cell growth. Or a similar medium is provided in the second compartment 80. A suitable medium may be any medium used to grow or maintain target cells. In order to create and maintain a larger colloid permeability compared to the medium of the first compartment 75 and / or the second compartment 80, a buffer containing a larger concentration of large molecules and / or larger polar molecules A third compartment 85 is provided. Materials used to create and maintain greater colloid penetration for the third compartment 85 include, but are not limited to, PEG 80000, albumin, other proteins or any other suitable material or solution. It is done. The membranes 90, 95 may have the same permeability or different permeability so that the waste from the growth of the medium and / or new cells is transferred to the first chamber 75 and Alternatively, the second chamber 80 may flow into the third chamber 85 by infiltration. The control system may maintain one or more volumes so that a constant volume is maintained in each compartment 75, 80, 85, a constant osmotic force is maintained, and a supply of fresh media to the second compartment 80 is maintained. Perfusion and drainage of media, buffer and / or waste from compartments 75, 80, 85 may be controlled. In addition, the control system by any suitable detection device, mechanism or method may be any suitable parameter involved in target cell growth, such as, but not limited to, changing the number of target cells, pH, CO ₂ , glucose Monitor calcium, potassium, sodium, temperature, humidity or any other suitable force, adjust the agitator movement interval, frequency and / or speed in the second chamber, and / or amount of medium, medium Any suitable adjustment based on the measurement of the control system to adjust the type of buffer, the amount of buffer, the type of buffer, the amount of CO ₂ or to improve or promote the growth of target cells in the chamber May be performed.

  Referring now to FIG. 4, another illustrative chamber 15 is shown. In this embodiment, the chamber may include one or more doorways 100 to provide a connection that allows fluid to flow between the chamber interior 35 and the waste container. The chamber 15 may further include one or more inlets / outlets 110 to provide a connection allowing fluid to flow between the chamber interior 35 and the cell culture medium container. Further, the chamber 15 may include one or more inlets / outlets 110 to provide a connection that allows fluid to flow between the chamber interior 35 and the buffer container. In addition, the chamber may further comprise one or more doorways 115 for receiving the agitator into the chamber interior 35. In one embodiment, the chamber 15 is made from Teflon® FEP. This specific embodiment would be useful when the target cells adhere to or otherwise connect to the agitator and waste is flushed from the chamber inside 35 and the target cells remain in the chamber inside 35. However, it will be appreciated that the chamber 15 may be made from any suitable material, and that is within the scope of the present disclosure.

The agitator 20 is disposed in the chamber inner side 35 and can move between the chamber first end 40 and the chamber second end 45. Alternatively, the agitator 20 may be configured to move between any two or more points, or between two or more parts, within the chamber interior 35. In the illustrative embodiment, the agitator includes a plurality of beads 21. Any illustrative embodiment showing the beads 21 may use any alternative agitator structure, which is also within the scope of this disclosure, and any specific illustrative embodiment is an agitator. It will be understood that the invention is not limited to the exclusive use of beads. In one embodiment, the beads 21 are made from a magnetizable material, such as silicone steel, Fe ₃ O ₄ , or any other suitable magnetizable material. As used herein, magnetizability refers to, for example, a magnetic field when an agitator (eg, a bead) retains a magnetic charge when exposed to a magnetic field, but when the magnetic field is removed from or near the agitator. This means that when the generator is turned off, it will not hold the magnetic charge in any other way. The magnetizable material typically includes a core for each bead 21. The magnetizable core may then be coated with any suitable material. In one embodiment, the magnetisable core is coated with polystyrene, but it will be understood that the magnetisable core may be coated with any suitable material, and that is within the scope of the present disclosure. For example, without limitation, the magnetizable core may be coated with any suitable thermoplastic or thermosetting polymer. Although the beads 21 are shown as being made from a magnetizable material, the beads may be made from any suitable material that is magnetic or non-magnetic, and still be within the scope of this disclosure. Will be understood. Further, it will be appreciated that the beads 21 may each be coated with any suitable material such that the target cells adhere to the beads as the cells grow in the chamber 15. Furthermore, it will be understood that beads that are not coated with a specific material to which the target cells will adhere also fall within the scope of the present disclosure. In certain embodiments, it may be desirable to include beads 21 that float in the cell culture medium. Thus, the core of the bead may comprise air pockets or bubbles, light bubbles or plastic or any other suitable material that allows the beads 21 to float in the medium.

  The beads 21 may be made such that when the beads are stacked, one or more niches or microenvironments may be formed or created in the voids between the beads 21. In certain embodiments, these niches may promote further target cell growth within the niche. In one embodiment, if the beads are substantially spherical, the diameter of each bead 21 may be 1 mm to 10 mm to create a suitable niche. However, it is understood that the beads 21 may have any suitable size and / or shape such that one or more suitable niches may be created when the beads 21 are stacked. It will be. Furthermore, it will be appreciated that at least some niche may be made between some beads inside the chamber and one or more walls.

  In an alternative embodiment, as shown in FIG. 5, the agitator 20a may be a planar member 21a having a plurality of openings 22 therein. The cross-section of the planar member 21a may be complementary to the cross-sectional shape of the chamber 15 so that the agitator 20a can move in the chamber inner side 35. The opening 22 may be such that the medium can flow through the agitator 20a as the agitator 20a moves through the chamber interior 35. The agitator 20a may be made from a magnetic or non-magnetic material, may be made to be floating or non-buoyant, and / or coated as described above for the beads 20. It may be.

  Referring again to FIG. 1, the control system 30 may include either or both of the controller 55 and the computer 60 to control the operation of the system 10. Alternatively, the system 10 may be moved manually. The control system 30 is configured to be detachably connected to the chamber 15. The control system 30 may include a cassette 50 that receives the chamber 15, but the chamber 15 is controlled by any suitable means or structure (eg, clip, hook, magnet, hook-loop assembly, friction fit, etc.). It will be understood that it may be connected to the system 50 and is within the scope of this disclosure.

The control system 30 further detects the number of cells in the light source 2 and the chamber 15, detects changes in the number of cells in the chamber 15, etc., and detects the results to the control system 30. A container 9 may be provided. However, it will be understood that any detector, mechanism or technique known in the art that monitors the number of cells or changes in the number of cells may be used and is within the scope of this disclosure. Let's go. In addition, the control system 30 with any suitable detector, mechanism or method may be used with any suitable parameters involved in target cell growth, such as, but not limited to, changes in the number of target cells, pH, CO ₂ , glucose Monitor calcium, potassium, sodium, temperature, humidity or any other suitable factor, adjust the frequency and / or speed of movement of the agitator in the chamber, and / or the amount of medium, type of medium, buffer the amount, type of buffer, to adjust the amount of CO _2, or by performing any appropriate adjustments based on the measurement of any of the control systems, such as one improving the growth of target cells in the chamber 15, or to promote Also good.

  The control system 30 is operable so that the agitator moves within the interior 35 of the chamber 15. This may be accomplished in a variety of ways. In the illustrative embodiment, the control system includes a motor 65 that is operable to rotate the chamber 15 between a first position and a second position. As described below, the first position and the second position are approximately 180 degrees apart, but the first and second positions may have any suitable angular relationship with each other. It will be understood that it is also within the scope of this disclosure. The chamber 15 may rotate in a horizontal plane, may rotate in a vertical plane, or rotate in any suitable manner, shift, slide, or otherwise move within the chamber 15 20 may move.

  In addition, the control system 30 includes a first magnetic field generator 70 and a second magnetic field generator 70 to excite the beads 21 or other agitator 20 so that the beads 21 in the chamber 15 move and mix the target cells and culture medium. A magnetic field generator 72 may be provided. In an illustrative embodiment, each magnetic field generator is an electromagnet that generates a magnetic field when turned on and stops generating a magnetic field when turned off. When the power is turned on, each magnetic field generator pulls the agitator 20 (for example, the beads 21) toward the magnetic field generator that is turned on. In an alternative embodiment, a permanent magnet may be used, in which case the control system 30 removes the magnet from the vicinity of the chamber 15 or otherwise applies a magnetic field from the magnet that has entered the chamber 15. Can be operated to shut off. If the illustrative embodiment uses chamber rotation and electromagnets to move the agitator within the chamber, it is understood that chamber rotation may be used alone or electromagnets may be used alone. Will be done. Further, it will be understood that any technique for moving the agitator within the chamber may be used and is within the scope of the present disclosure.

  2a-2f, the operation of system 10 is illustrated by a non-limiting example. Target cells and cell culture are carried into the interior 35 of the chamber 15. In this embodiment, the beads 21 are buoyant and float near the top of the cell culture medium in the chamber 15. In FIG. 2 a, the first magnetic field generator 70 is turned on and the beads 21 are held near the first end 40 of the chamber 15. The chamber 15 is then rotated approximately 180 ° to the position as shown in FIG. 2b, and the first magnetic field generator 70 maintains the beads 21 near the first end 40 of the chamber. The first magnetic field generator 70 is then turned off, causing the beads 21 to begin to float toward the second end 45 of the chamber 15, as shown in FIG. In embodiments where the beads 21 include a coating to which the target cells adhere, movement of the beads 21 from one end to the other will cause newly grown target cells to collect. As waste flows from chamber 15 and / or when new media is introduced into chamber 15, target cells are transferred to beads 21 such that a significant amount of the original and newly grown target cells remain in the chamber. It may be adhered. Alternatively, by adding a magnetizable antibody specific to the target cell to the inside of the chamber 15, when the antibody binds to the target cell and a magnetic field is introduced into the chamber, the target cell to which the antibody has bound is magnetized. It will removably connect to the chamber wall adjacent to the beads 21 and / or the magnetic field generator. In this embodiment, fresh media is used so that unbound cells and / or waste are flushed from the chamber and / or significant amounts of original and newly grown target cells remain in chamber 15. May be left on while either or both of the magnetic field generators 70, 72 are turned on. Alternatively, a magnetic reagent (eg, Annexin V or other suitable reagent) may be used to connect the beads to damaged or dead cells shed from the system 10, healthy target cells. Further, magnetizable antibodies and / or reagents may be used in chamber 15 without the use of an agitator so that when the chamber is flushed, target cells or damaged / dead cells are It will be understood that it may be held on.

Referring again to the figure, if the bead 21 is near the second end 45 of the chamber, the bead 21 is held near the second end 45 of the chamber by turning on the second magnetic field generator 72 (see FIG. 2d) The chamber rotates to the position shown in FIG. 2e. The second magnetic field generator 72 is then turned off, causing the beads 21 to float toward the first end 40 of the chamber, as shown in FIG. 2f. As will be appreciated by those skilled in the art, this process various additives in the culture medium, a buffer may be optionally added at any given point and the chamber CO _2, and / or previously as described Waste may be selectively removed to promote or improve new cell growth based on measurements made by the control system.

  In an alternative embodiment, non-buoyant beads may be used without additional magnetic fields so that the beads move through the chamber as the chamber rotates. Here, the beads move between two or more points in the chamber 15 using gravity and centrifugal force due to the rotation of the chamber. In yet another alternative, the power supply of the first magnetic field generator 70 and the second magnetic field generator 72 without rotating the chamber 15 so that the beads move between two or more points in the chamber. May be inserted alternately. While the above example uses beads 21 as an agitator, it is understood that any suitable device may be used as an agitator, including but not limited to that of FIG. 5, which is also within the scope of this disclosure. right. Further, it will be understood that any means or technique for moving the agitator within the chamber may be used and is within the scope of the present disclosure.

Further, it is understood that if the chamber 15 is made from a gas permeable material or otherwise includes a gas permeable portion, the system may be located in a CO ₂ incubator or CO ₂ chamber. Will be done. If there is no CO ₂ incubator or CO ₂ chamber, or there is no gas permeable portion in the chamber, a reagent (eg, HEPES) may be used, or CO ₂ is injected directly from the CO ₂ container into the chamber. Also good.

  Although the invention and its advantages have been disclosed in terms of specific examples, non-limiting embodiments, various modifications and substitutions have been made without departing from the scope of the invention as defined by the appended claims. It should be understood that replacements and modifications can be made. It will be understood that any feature described in conjunction with any one embodiment may be applied to other embodiments.

Claims (33)

A bioreactor system for separating cells based on cell markers and growing cells and artificial tissue,
One reaction chamber comprising an interior having a first part and a second part, and providing a space for the reaction of cell separation by cell markers, cell growth and artificial tissue growth;
A magnetizable agitator disposed inside the chamber and movable between an inner first portion and an inner second portion;
Operates to assist cell separation with cell markers, allowing the agitator to stay in a specific inner position of the chamber, or to move between the inner first part and the inner second part as the reaction chamber inverts A bioreactor system comprising at least an adjustable magnetic field. The inside of the reaction chamber is
A first compartment (low permeation zone), coupled to the fluid flow of the cell culture medium container, for receiving and erasing the cell culture medium from the container, comprising a low colloid osmotic solution;
A second compartment (reaction compartment) that is adjacent to the first compartment and that is designed to serve as an entrance / exit for receiving and erasing cells, agitators, reagents, buffers and media, and in which the agitator is placed. )When,
A first membrane disposed between the first compartment and the second compartment and made to allow selective flow of cell culture medium from the first compartment to the second compartment;
Adjacent to the second compartment, the third compartment is coupled to allow fluid to and from the buffer container so that the buffer can receive the buffer from the buffer container, and the waste is disposed from the third container to the waste container. A third compartment (high permeation compartment) designed to connect the waste container and fluid so that it can flow to
A second membrane disposed between the second compartment and the third compartment and made to allow selective flow of media and waste from the second compartment to the third compartment;
At least part of the side wall of the reaction chamber made of a gas permeable material, one component of a medium containing a reagent for maintaining pH (eg HEPES), or a gas permeable system connected to the chamber, or these 3 Any combination of
The bioreactor system of claim 1, comprising a bioreactor cassette and at least a portion of a reaction chamber located in an optional holder. A magnetic adherent agitator comprising a planar member having a plurality of beads or a plurality of openings,
(1) Made entirely of rigid magnetizable material, bare (uncoated), or inert, rigid or rigid for any purpose including cell attachment and cell damage reduction Or (2) at least partially magnetic when placed in a magnetic field, but becomes non-magnetic or magnetic when the magnetic field disappears or is removed from the reaction zone Made of magnetically adherent material that makes it very weak,
(3) The bioreactor system of claim 1, wherein the beads have a diameter greater than 1 millimeter and less than the volume of the reaction chamber of claims 1 and 2.   The magnetic adherent agitator binds to antibodies or other biomolecules labeled with a magnetic adherent material to attach and grow cells in adherent cell cultures and during cell separation with cell markers 4. The bioreactor system of claim 3, which functions as a cell adjunct to connect cells and includes biomolecule reaction and binding and cell capture and release controlled by a magnetic field.   Using the magnetized agitator, the medium is slowly mechanically agitated by moving between any two parts in the reaction chamber, (1) the attractive force of the surrounding magnetic field, (2) the agitator is in the reaction chamber The cells are suspended in the liquid growth medium by a mechanism such as buoyancy when the density is lower than the medium of the above, or (3) gravity when the beads are higher than the medium in the reaction chamber. The bioreactor system according to claim 3.   The magnetizable agitator is used to create a “niche” or microenvironment for cell growth, where the niche moves each by the mechanism described in claim 5 as the magnetizable agitator precipitates. The bioreactor of claim 1, comprising a space between the agitators to prevent any excessive crowding or agglomeration that can interfere with cell growth and reduce non-specific differentiation of stem cells.   (Delete) The adjustable magnetic field,
(1) Can be made by an electromagnet that adjusts the magnetic field by changing the current and / or voltage, or by changing the position and orientation (orientation) of the electromagnet,
(2) It can be made by a permanent magnet that changes the magnetic field by changing the position and orientation of the permanent magnet, or (3) it can be made by a combination of (1) and (2) above The bioreactor according to 1.   A bioreactor system for separating cells and growing cells and artificial tissue comprising a protective perfusion system, the protective perfusion system further comprising one or more reaction chambers and one or more between the compartments. Dialysis membrane wall, osmotic buffer, gradient osmotic buffer container, and associated dynamic device for containers that circulate the buffer by osmosis and collect waste, from the low-permeability compartment, dialyze New medium can flow through the membrane to the reaction compartment (or a second compartment as described in claim 2), while the old medium from the reaction compartment is removed before being discarded into the waste container. A bioreactor system that flows through a dialysis membrane to a high osmotic compartment.   The gradient osmotic buffer includes a high concentration of large molecules, e.g., a high colloid osmotic buffer containing protein and PEG 8000 to create a high colloid osmotic force, and a small amount of large molecules. 10. The bioreactor of claim 9, comprising a low colloid osmotic buffer free of any.   Bioreactor magnetic field, perfusion, reaction chamber inversion and agitator movement can be automatically adjusted using preselected programs or based on signals or information obtained by detectors and sensors from the reaction environment 10. The bioreactor system of claim 1 and 9, further comprising a control system comprising a computer programmed and feedback system.   Prepare cells for cell separation with cell markers, isolate target cells in bioreactors, discard unselected cells and old media, and real-time cell density and cell growth conditions Program and apply a computerized system to adjust and optimize cell culture conditions for real-time monitoring, cell growth and direct differentiation, and to adjust and optimize cell culture conditions Separating cells based on cell markers comprising: culturing cells in a culture chamber with or without a niche, and collecting cells in a bioreactor; and How to grow a population organization.   Separating the cells based on the cell marker and growing the cells is performed in the same reaction chamber, and the cell separation by the cell marker can be performed before, after and during cell culture, Item 13. The method according to Item 12.   (Delete)   (Delete)   13. The method of claim 12, wherein monitoring cell density in real time can be performed using optical and / or biochemical principles.   Growing cells can be controlled by the control system if the bioreactor keeps the cells in suspension or standing, or in a program that alternates between suspension and standing. 13. The method of claim 12, wherein the method is controlled and performed by combining mechanical agitation with an agitator of the chamber vessel and cell culture.   The cells are kept in suspension by movement of a magnetized agitator, and this movement is controlled by an adjustable magnetic field with or without assistance from gravity and / or buoyancy. Item 18. The method according to Item 17.   The magnetizable agitator controlled by an adjustable magnetic field can be moved in any direction between any two parts inside the reaction chamber, this movement being assisted by gravity and / or buoyancy. 19. A method according to claim 18, wherein movement along the vertical axis is preferred, controlled by an adjustable magnetic field with or without assistance.   13. The niche is formed in a magnetic adherent agitator when the magnetic adherent agitator is physically stationary and can be adjusted by moving the magnetic adherent agitator. Method.   13. The method of claim 12, wherein the control system is programmable and programmed for all or part of a bioreactor function.   Real-time adjustment and optimization of cell culture conditions for cell growth and direct differentiation is controlled by a computer including a cell culture condition detector, computer or similar device and corresponding program, feedback operation control system The method of claim 12, wherein the method is performed by a combined complex system.   Cell culture conditions for direct differentiation are for all cells with differentiation potential, including but not limited to all types of stem cells, and the direct differentiation can induce hepatocytes and their blood vessels simultaneously, for example. 13. The method of claim 12, wherein two or more induction directions can be induced simultaneously by biochemical and physical methods.   Bioreactors with reversed current include but are not limited to hematopoietic stem cells, mesenchymal stem cells, fibroblasts, hybridoma cells, lymphocytes, dendritic cells, insect cells, embryonic stem cells, various tissue cells, cancer cells and cell lines, The bioreactor of claim 1, capable of providing ideal cell growth conditions for all cells, including transformed cells and cell lines.   26. The protective perfusion system of claim 9 and the chamber of claim 25, which can be used individually or in conjunction with cell culture, cell separation and other techniques in any field that would benefit from these techniques. Bioreactor systems combining inversion and mechanical stirring of solutions and related methods thereof.   A bioreactor system for separating cells and growing cells and artificial tissue, controlled by an adjustable magnetic field in claims 7 and 8 or controlled by a control system in claim 11 A bioreactor system comprising a reaction chamber and an agitator capable of holding cells and agitator in suspension by combining chamber inversion and mechanical agitation of the agitator.   After applying a combination of inversion of the reaction chamber and mechanical stirring of the cells for cell suspension, after starting with static culture, so that the cells are in the best growth conditions for both static and dynamic culture, 26. The bioreactor system according to claims 1 and 25, wherein the bioreactor system is capable of culturing dynamically and distributing the cells uniformly within the reaction compartment.   Growing cells and tissue is performed in a protective perfusion chamber where a perfusion solution containing small molecules passes through the membrane in a single direction from the low osmosis compartment to the reaction compartment and then to the high osmosis compartment. 10. The bioreactor system of claim 9, wherein this passage occurs for a colloid osmotic gradient but does not have to follow a concentration gradient of small molecules in different compartments as occurs in general dialysis.   10. The bioreactor system of claim 9, wherein the protective perfusion system can be used for rapid media exchange, such as media depletion and media addition, and cell suspensions that do not lose cells, peptides and proteins.   12. The bioreactor system of claim 1, further comprising a protective perfusion system as in claim 9 and a control system as in claim 11 for separating cells based on cell markers and growing cells and artificial tissue. .   2. The bio of claim 1, wherein the adjustable magnetic field can be adjusted by changing the strength, direction or position of the magnetic field by a control system during cell separation by cell markers, cell growth and artificial tissue growth. reactor. Cell separation by cell marker
Binding a specific magnetized antibody or other biomolecule to a target cell;
9. Capture cells magnetically labeled by either a magnetizable agitator or chamber wall, or by both the magnetizable agitator and chamber wall, wherein the chamber wall is by the generator of claim 8 (magnet or electromagnet). Made to adjust the magnetic field,
Discarding or washing unlabeled cells;
The method of claim 13, comprising releasing target cells.   Cell separation by cells is performed in the reaction compartment of the chamber (second compartment as described in claim 2), and a biological reaction using a biomolecule that specifically binds to a biological marker of the cell, 35. The method of claim 32, wherein the method is performed by a procedure comprising washing suspended cells.

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